多种PEG化重组人生长激素中蛋白含量测定的通用型方法的建立

Establishment of a general method to determine the protein content of various PEG-rhGHs

目的：通过以未修饰的原型蛋白为对照品,建立准确测定多种聚乙二醇化重组人生长激素（PEG-rhGH）中蛋白含量的通用型方法。方法：采用rhGH国家对照品和凝胶色谱法测定PEG-rhGH中的蛋白含量。通过详细的专属性、精密度、准确性等方法学考察,建立该方法。色谱条件：采用TSKGel 3000SWxl（300 mm×7.8mm）凝胶色谱柱,以0.05 mol·L-1的磷酸氢二钠-磷酸二氢钠（pH7.0）为流动相,流速为0.6 mL·min-1,柱温为室温,检测波长为280 nm。通过将测定结果与申报企业原有方法测定结果进行比对,对现有含量测定方法进行评价。结果：由于PEG与蛋白质之间的连接子存在双键,在214 nm下,3种PEG-rhGH的测定结果的准确性较差,平均回收率（n=9）分别为109.3%、109.9%、110.6%;而在280 nm下,rhGH在200-1800μg·mL-1浓度范围内线性关系良好（r=0.9999）,3种PEG-rhGH的平均回收率（n=9）分别为101.2%、101.7%、100.4%。结果比对中发现,新建方法与企业申报方法中采用同质对照品的HPLC法测定结果相当,但与以BSA为对照品的Lowry法测定结果相差较大。结论：本方法专属性、精密度和准确性良好,可作为多种PEG-rhGH含量测定的通用型方法。

Objective：To develop a general assay method for various pegylated recombinant human growth hormones（ PEGrhGHs） by using unmodified protein as a reference substance. Methods：A national standard,rhGH,and a gel permeation chromatography（ GPC） method were used to determine the protein content of PEGrhGHs.This method was well established by validation on the specificity,precision and accuracy. The assay was performed on a TSKGel 3000 SWxl column（ 300 mm × 7. 8 mm） at room temperature with the mobile phase of 0. 05 mol·L1Na2HPO4NaH2PO4（ pH 7. 0）. The flow rate was 1.0 mL·min1and the detection wavelength was set at 280 nm. Current assay methods were evaluated through result comparison between this general method and those methods submitted by applicant companies. Results：Because there were double bonds in the linker between PEG and protein,the accuracy for the three kinds of PEGrhGHs was poor at 214 nm. The average recoveries were 109. 3%,109. 9%,110. 6%,respectively. However,when the wavelength was changed to 280 nm,rhGH showed a good linear relationship（ r = 0. 9999） in the range of 2001800 μg·mL1and the average recoveries of the three different PEGrhGHs were 101. 2%,101. 7%,100. 4%,respectively. Result comparison showed that contents obtained by the new method were similar with those obtained by HPLC methods using homogeneous standards but quite different from the result obtained through Lowry method using BSA as a standard. Conclusion：The method is specific,precise and accurate,and can be used as a general assay for various PEGrhGHs.